Abstract Cell mechanics has been increasingly found to play a significant role in all aspects of cell function, including signaling, migration, differentiation, and survival. The altered state of stress-bearing elements in cells occurs during malignant transformation and metastasis, in hemolytic anemia, nephrotic syndrome, and muscular dystrophy. The distribution of stress and strain in cells is complex and the associated feedback mechanisms are not well understood. Progress in single-molecule biophysics and synthetic biology led to the development of FRET-based molecular sensor domains reporting on local forces in the cytoskeleton and ECM. However, there is essentially no information about tension in the plasma membrane and no principle for its measurement has been proposed to date. Our recent study of the mechanosensitive channel blocker Grammostola mechanotoxin (GsMTx4) indicated that the peptide binds to the membrane but undergoes a sharp redistribution between the ?shallow? and ?deep? binding modes in a narrow range of membrane tensions near the resting tension of the membrane. The change in membrane area contributed by the inserted peptide produces a ?tension clamp? effect in one leaflet and redistribution of tension to the other. In this proposal we will explore this principle of the tension-dependent transition of this conical amphipathic molecule in the membrane to create a set of fluorescent tension reporters based on the GsMTx4 peptide scaffold. Aim 1 will include computational design of a synthetic sensor peptide equipped with an environmentally-sensitive fluorescent sidechain in a specific location and experimental calibration of its fluorescent signal in model membranes. The tests will include measurements of fluorescence as a function of lateral pressure/tension in Langmuir monolayers, pipette aspiration experiments with giant unilamellar vesicles and cytoskeleton- depleted membrane blebs, and osmotic shock of liposomes in stopped-flow experiments. These tests will enable optimization of peptide sensitivity in specific tension ranges. Aim 2 will validate the use of GsMTx4- based sensors in live cells using a range of mechanical stimuli. Membrane stress in cells will be generated using blunt probes, stretch on an elastic support, osmotic shock, fluid shear stress, and by motor proteins as cells adhere and move on substrates. We will look for heterogeneities in GsMTx4 surface distribution, and global and local responses to tension application and release. We will determine if stressed (brighter) or protected regions co-localize with ordered domains, focal adhesions, processes and invaginations, or mechanosensory complexes. Fluorescent signals of the surface-bound tension sensors will be correlated with activities of mechanosensitive channels (Piezo 1) under various mechanical stimuli. We will compare the kinetics of the membrane tension changes to changes in cytoskeletal stress using our FRET based fluorescent force sensor cpstFRET. The optimized set of sensors will become a tool for quantification of membrane tension, which remains a missing link in many normal signaling mechanisms and in pathology.